Bottom Line:
Modification while channels were opening and closing in the presence of ATP caused macroscopic CFTR current to decline at the same speed as when the unmodified channels shut upon sudden ATP withdrawal.We conclude that, in every CFTR channel gating cycle, the NBD dimer interface separates simultaneously at both composite sites sufficiently to allow MTS reagents to access both signature-sequence serines.Relatively rapid modification of S1347C channels by larger reagents-MTS-glucose, MTS-biotin, and MTS-rhodamine-demonstrates that, at the noncatalytic composite site, this separation must exceed 8 Å.

Mentions:
S549 in the LSGGQ sequence of the NBD1 tail contributes to CFTR’s catalytically competent composite site and, by homology with nucleotide-bound NBD homodimer crystal structures, is expected to hydrogen bond to the γ phosphate of the ATP held by the NBD2 Walker A motif (Hopfner et al., 2000; Smith et al., 2002; Chen et al., 2003). To assess accessibility of introduced S549C, we applied the small hydrophilic, sulfhydryl-specific MTS reagent MTSET+ (50 µM; Fig. 3 A) to S549C-(C832S-C1458S) CFTR channels opening and closing in inside-out patches exposed to 3 mM MgATP (Fig. 3 A). Despite the maintained presence of ATP, the MTSET+ caused a rapid current decline (Fig. 3 A). The current decay reflects modification of the introduced cysteine because background (C832S-C1458S) CFTR channels, lacking the engineered target cysteine, are little affected (Fig. S2 A) by much higher concentrations of MTSET+ or of the similarly sized MTS reagents, negatively charged MTSES−, or neutral MTSACE; in these control (C832S-C1458S) CFTR channels, all eight native cysteines in the C-terminal half of CFTR have been replaced by serines, but all 10 native N-terminal cysteines remain (compare Mense et al., 2006). Near abolition of CFTR current by MTSET+ while ATP was present indicates that CFTR channels with a covalently attached MTSET+ adduct at position 549 can essentially no longer be opened by ATP. This corroborates impaired activity of S549C CFTR after permanent attachment of similarly sized (Fig. 2), but neutral, NEM (Cotten and Welsh, 1998).

Mentions:
S549 in the LSGGQ sequence of the NBD1 tail contributes to CFTR’s catalytically competent composite site and, by homology with nucleotide-bound NBD homodimer crystal structures, is expected to hydrogen bond to the γ phosphate of the ATP held by the NBD2 Walker A motif (Hopfner et al., 2000; Smith et al., 2002; Chen et al., 2003). To assess accessibility of introduced S549C, we applied the small hydrophilic, sulfhydryl-specific MTS reagent MTSET+ (50 µM; Fig. 3 A) to S549C-(C832S-C1458S) CFTR channels opening and closing in inside-out patches exposed to 3 mM MgATP (Fig. 3 A). Despite the maintained presence of ATP, the MTSET+ caused a rapid current decline (Fig. 3 A). The current decay reflects modification of the introduced cysteine because background (C832S-C1458S) CFTR channels, lacking the engineered target cysteine, are little affected (Fig. S2 A) by much higher concentrations of MTSET+ or of the similarly sized MTS reagents, negatively charged MTSES−, or neutral MTSACE; in these control (C832S-C1458S) CFTR channels, all eight native cysteines in the C-terminal half of CFTR have been replaced by serines, but all 10 native N-terminal cysteines remain (compare Mense et al., 2006). Near abolition of CFTR current by MTSET+ while ATP was present indicates that CFTR channels with a covalently attached MTSET+ adduct at position 549 can essentially no longer be opened by ATP. This corroborates impaired activity of S549C CFTR after permanent attachment of similarly sized (Fig. 2), but neutral, NEM (Cotten and Welsh, 1998).

Bottom Line:
Modification while channels were opening and closing in the presence of ATP caused macroscopic CFTR current to decline at the same speed as when the unmodified channels shut upon sudden ATP withdrawal.We conclude that, in every CFTR channel gating cycle, the NBD dimer interface separates simultaneously at both composite sites sufficiently to allow MTS reagents to access both signature-sequence serines.Relatively rapid modification of S1347C channels by larger reagents-MTS-glucose, MTS-biotin, and MTS-rhodamine-demonstrates that, at the noncatalytic composite site, this separation must exceed 8 Å.